Microarray-based Gene Expression Profiling Research Articles

Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM. Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed inThe Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3'UTR luciferase assays. The effect of miR-196a overexpression/inhibition wasassessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies wereperformrd to study the effect of miR-196a ontheGBM cellular transcriptome under hypoxia. Weidentified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196ayielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a inHIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs andin maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia. Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Suppression of microRNA-323-3p restrains vascular endothelial cell apoptosis via promoting sirtuin-1 expression in coronary heart disease

AimsCoronary heart disease (CHD), a chronic inflammatory condition of vascular endothelial cells (VECs), poses a serious threat to human health. Previous studies have found that microRNAs (miRNAs) are closely related to the occurrence and development of cardiac diseases. Therefore, this study focused on the regulation by miR-323-3p on the progression of CHD. MethodsInitially, we employed microarray-based gene expression profiling of CHD to identify differentially expressed miRNAs. Next, the expression of miR-323-3p and SIRT1 was detected by RT-qPCR in a rat model of CHD generated by feeding with a high-fat diet. The interaction between miR-323-3p and SIRT1 was identified using bioinformatics analysis and dual luciferase reporter gene assay. The expressions of miR-323-3p and SIRT1 were altered in CHD rats and vascular endothelial cells (VECs) to examine the specific effects on CHD. ResultsmiR-323-3p was observed to be highly-expressed in blood samples from patients with CHD or with mild atherosclerosis and in the rat model of CHD. SIRT1 was a target gene of miR-323-3p, which could downregulate SIRT1 expression. miR-323-3p overexpression or SIRT1 inhibition resulted in increased apoptosis of VECs, elevated ac-p65 protein expression and ratio of ac-p65/p65, and upregulated expression of NF-κB signaling pathway-related proteins. Besides, miR-323-3p inhibition or SIRT1 upregulation in the CHD rat model was found to significantly alleviate symptoms and decrease levels of proteins related to the ac-p65 and NF-κB signaling pathways. ConclusionOverall, the experimental data provide evidence that miR-323-3p suppression may restrain VEC apoptosis and prevent the resultant CHD progression via SIRT1-inactivatedNF-κB signaling pathway.

Gene expression in blood reflects smoking exposure among cancer-free women in the Norwegian Women and Cancer (NOWAC) postgenome cohort

Active smoking has been linked to modulated gene expression in blood. However, there is a need for a more thorough understanding of how quantitative measures of smoking exposure relate to differentially expressed genes (DEGs) in whole-blood among ever smokers. This study analysed microarray-based gene expression profiles from whole-blood samples according to smoking status and quantitative measures of smoking exposure among cancer-free women (n = 1708) in the Norwegian Women and Cancer postgenome cohort. When compared with never smokers and former smokers, current smokers had 911 and 1082 DEGs, respectively and their biological functions could indicate systemic impacts of smoking. LRRN3 was associated with smoking status with the lowest FDR-adjusted p-value. When never smokers and all former smokers were compared, no DEGs were observed, but LRRN3 was differentially expressed when never smokers were compared with former smokers who quit smoking ≤ 10 years ago. Further, LRRN3 was positively associated with smoking intensity, pack-years, and comprehensive smoking index score among current smokers; and negatively associated with time since cessation among former smokers. Consequently, LRRN3 expression in whole-blood is a molecular signal of smoking exposure that could supplant self-reported smoking data in further research targeting blood-based markers related to the health effects of smoking.

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients. MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database. ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome. ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.

DPP4 gene silencing inhibits proliferation and epithelial-mesenchymal transition of papillary thyroid carcinoma cells through suppression of the MAPK pathway.

Papillary thyroid carcinoma (PTC) is characterized by epithelial malignancy and is the most prevalent thyroid neoplasmwith the best overall prognosis. Notably, recently published studies have indicated remarkably high expression of dipeptidyl peptidase 4 (DPP4) in PTC. However, the underlying molecular mechanism and regulatory factors of PTC progression remain unknown. Therefore, the current study aimed to elucidate the effects of DPP4 gene silencing on PTC and further investigated whether the mechanism of PTC progression is related to the mitogen-activated protein kinase (MAPK) pathway. Herein, microarray-based gene expression profiling of PTC was conducted to identify the differentially expressed genes between tumor thyroid tissue and normal thyroid tissue as well as the underlying signaling pathway involved in PTC pathogenesis. Moreover, protein quantification was performed to assess the protein expression of DPP4 in PTC tissues collected from 65 patients. In addition, DPP4 was silenced in PTC cell lines (GLAG-66 and TPC-1) through siRNA-mediated DPP4 knockdown or sitagliptin (inhibitor of DPP4)-mediated inhibition to assess the effects of DPP4 on the MAPK pathway and cellular processes, including proliferation, apoptosis, and epithelial-to-mesenchymal transition (EMT). Intriguingly, our data revealed markedly high expression of DPP4 in PTC tissues. However, in GLAG-66 and TPC-1 cells, the silencing of DPP4 resulted in significantly reduced expression of ERK1/2, JNK1, P38 MAPK, VEGF, FGFR-1, TGF-β1, Snail, HIF-1α, N-cadherin, and Bcl-2 along with reduced phosphorylation of ERK1/2, JNK1, and P38 MAPK, whereas the expression of E-cadherin and Bax was increased. Furthermore, DPP4 silencing was found to hinder cell proliferation and potentiate cell apoptosis. Collectively, the present study demonstrated that DPP4 gene silencing inhibits PTC cell proliferation and EMT and promotes cell apoptosis via suppression of the MAPK pathway, thus highlighting a possible regulatory pathway in PTC progression.

Microarray based gene expression profiling of advanced gall bladder cancer.

Gall bladder cancer (GBC) is an aggressive cancer with specific predilection like female gender and specific geographical areas, however the molecular mechanisms and factors contributing to the clinical or biological behavior are not understood. The aim of this study was to perform a comprehensive analysis of differentially expressed genes in advanced GBC and chronic cholecystitis(CC) cases. Microarray was planned on fresh specimens of advanced GBC and CC cases using single color cRNA based microarray technique (8X60K format; Agilent Technologies, USA). Twelve advanced GBC and four CC patients were included in the study. Of the total of 1307differentially expressed genes, 535genes were significantly upregulated, while 772genes were significantly downregulated in advanced GBC vs CC samples. Differentially expressed genes were associated with biological processes (55.03%), cellular components (31.48%), and molecular functions (13.49%) respectively. The important pathways or key processes affected were cell cycle, DNA replication, oxidative stress, gastric cancer pathway. Using in silico analysis tools, three differentially expressed genes i.e. TPX2, Cdc45and MCM4were selected (for their significant role in DNA replication and microtubule function) and were further validated in 20advanced GBC cohort by immunohistochemistry. Significant positive association of Cdc45and MCM4proteins was found in advanced GBC cases (p=0.043), suggesting the probable oncogenic role of Cdc45and MCM4proteins in advanced GBC. Our data demonstrate the potential regulation of Cdc45-MCM4axis in advanced GBC tumors. Additionally, our study also revealed a range of differentially expressed genes (e.g. TPX2, AKURA etc.) between GBC and CC, and further validation of these genes might provide a potential diagnostic or therapeutic target in future.

LncRNA MAGI2-AS3 Overexpression Sensitizes Esophageal Cancer Cells to Irradiation Through Down-Regulation of HOXB7 via EZH2.

BackgroundAccumulating evidence has suggested that aberrant expression of long non-coding RNAs (lncRNAs) may contribute to cancer progression in association with radioresistance. The current study aimed to identify the potential role of lncRNA MAGI2-AS3 and the underlying mechanism in its regulation of the radio-sensitivity of esophageal cancer cells.Methods and ResultsInitially, we detected high expression of HOXB7 from microarray-based gene expression profiling of esophageal cancer. Then, we identified the interactions among MAGI2-AS3, HOXB7, and EZH2 by dual-luciferase reporter gene assay, RNA pull-down assay, RIP assay and ChIP assay. HOXB7 was highly-expressed, while MAGI2-AS3 was poorly-expressed in esophageal cancer tissues and cells. The effect of MAGI2-AS3 and HOXB7 on esophageal cancer cell proliferation and apoptosis as well as tumorigenicity of radioresistant cells was examined by gain- and loss-of-function experiments. Interestingly, MAGI2-AS3 down-regulated HOXB7 through interaction with EZH2, which promoted cell apoptosis and inhibited proliferation and radio-resistance. Besides, down-regulation of MAGI2-AS3 exerted a promoting effect on these malignant phenotypes.ConclusionTaken together, our results reveal the potential role of MAGI2-AS3 over-expression in controlling esophageal cancer resistance to radiotherapy by down-regulating HOXB7, this providing a candidate biomarker for resistance to radiotherapy.

Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1.

Evidence has documented the tumor-promoting properties of long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in many cancers. However, little is known about its role in gallbladder cancer. Here, we aimed to characterize the functional relevance of lncRNA FOXD2-AS1 in gallbladder cancer and the possible mechanisms associated with methylation of MutL homolog-1 (MLH1). Initially, microarray-based gene expression profiling of gallbladder cancer was employed to identify differentially expressed lncRNAs. Next, the expression of lncRNA FOXD2-AS1 was examined, and the cell line presenting with the highest lncRNA FOXD2-AS1 expression was selected for subsequent experimentation. Then, the interaction between lncRNA FOXD2-AS1 and MLH1 was identified. The effect of lncRNA FOXD2-AS1 on proliferation, migration, invasion, and apoptosis as well as tumorigenicity of transfected GBC-SD cells was examined with gain- and loss-of-function experiments. We found that lncRNA FOXD2-AS1 was highly expressed, while MLH1 was poorly expressed in gallbladder cancer cells. Besides, lncRNA FOXD2-AS1 could promote MLH1 methylation by recruiting DNMT1 to the MLH1 promoter, and consequently inhibit MLH1 transcription. Silencing of lncRNA FOXD2-AS1 or overexpression of MLH1 inhibited gallbladder cancer cell proliferation, invasion, and migration, while facilitating cell apoptosis in vitro as well as retarding tumor growth in vivo. Thus, silencing of lncRNA FOXD2-AS1 suppressed the progression of gallbladder cancer via upregulation of MLH1 by inhibiting MLH1 promoter methylation. These findings present lncRNA FOXD2-AS1 knockdown as a potential candidate for the treatment of gallbladder cancer.

Abstract 571: Andrographolide enhances sensitivity to 5-fluorouracil by targeting ferroptosis-related genes in colorectal cancer

Abstract Purpose: Despite improvements in response rates from various treatment strategies, the 5-year survival rates for metastatic colorectal cancer (mCRC) remain dismal (~10-15%). One of the primary reasons for CRC therapy failure is development of acquired drug resistance to first line chemotherapies such as 5-fluorouracil (5FU), and nearly half of all mCRC patients are resistant to 5FU-based therapies. These limitations highlight the need for novel treatment strategies that offer improved efficacy and can help overcome acquired chemoresistance and enhance anti-cancer efficacy of conventional chemotherapies. Considering that some naturally-occurring botanicals have chemo-sensitizing properties, we analyzed the potential of andrographolide, a botanical with key biological activities, including anti-inflammatory, antitumorigenic, apoptotic, antiangiogenic and immunomodulatory activities, in reversing chemoresistance in CRC, and also delineated the associated molecular pathways underpinning this activity. Experimental design: We performed a series of in-vitro and in-vivo experiments to investigate the chemo-sensitizing ability of andrographolide in 5-FU resistant (5FUR) CRC cells, xenografts animal models and CRC-derived organoids. Furthermore, we performed gene expression microarrays and other mechanistic studies to explore the molecular mechanisms responsible for its chemo-sensitizing activity. Results: Andrographolide treatment significantly enhanced the efficacy of 5FU in HCT116 5FUR and SW480 5FUR cells, as evidenced by enhanced apoptosis and decreased proliferation rates (p<0.05). In line with our in-vitro findings, treatment with andrographolide also led to significant tumor growth inhibition in a xenograft animal model (p<0.0001). Likewise, in accordance with the results from cell lines and mice xenografts, the combination of 5FU and andrographolide significantly decreased the growth of patient-derived tumor organoids compared to those treated with 5FU alone (p<0.01 for patient 1, p<0.05 for patient 2). Through microarray-based gene expression profiling analysis in 5FU-resistant CRC cells, we identified ferroptosis and WNT signaling as the key cancer associated pathways responsible for andrographolide mediated chemo-sensitization in CRC cells. Taken together, using genome-wide gene expression analysis, we delineated the mechanism underlying the chemo-sensitizing properties of andrographolide and made a case for the potential use of andrographolide for enhancing the therapeutic efficacy of 5FU in CRC. Conclusion: Our data provides first evidence for andrographolide mediated sensitization to 5FU based therapy, through modulation of ferroptosis pathway in CRC. These results highlight the potential adjunctive therapeutic role for andrographolide in combination with conventional chemotherapeutic drug in patients with CRC. Citation Format: Priyanka Sharma, Tadanobu Shimura, Jasjit K Banwait, Ajay Goel. Andrographolide enhances sensitivity to 5-fluorouracil by targeting ferroptosis-related genes in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 571.

Exercise-mediated downregulation of MALAT1 expression and implications in primary and secondary cancer prevention

Long non-coding RNAs (lncRNAs) play critical roles in various biological functions and disease processes including cancer. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was initially identified as a lncRNA with elevated expression in primary human non-small cell lung tumors with high propensity to metastasize, and subsequently shown to be highly expressed in numerous other human cancers including breast, ovarian, prostate, cervical, endometrial, gastric, pancreatic, sarcoma, colorectal, bladder, brain, multiple myeloma, and lymphoma.MALAT1 is deeply involved in several physiological processes, including alternative splicing, epigenetic modification of gene expression, cellular senescence, healthy aging, and redox homeostasis.The aim of this work was to investigate the modulation exerted by a single bout of endurance exercise on the level of MALAT1 expression in peripheral blood mononuclear cells (PBMCs) from healthy male donors displaying different training status and redox homeostasis features. Our findings show that MALAT1 is downregulated after acute endurance exercise in subjects whose fitness level guarantee a high expression of SOD1 and SOD2 antioxidant genes and low levels of endogenous oxidative damage. In vitro protocols in Jurkat lymphoblastoid cells exposed to pro-oxidant environment confirmed the link between MALAT1 expression and antioxidant gene modulation, documenting p53 phosphorylation and its recruitment to MALAT1 promoter. Remarkably, analyses of Microarray-Based Gene Expression Profiling revealed high MALAT1 expression in leukemia patients in comparison to healthy control and a significant negative correlation between MALAT1 and SOD1 expression.Collectively our results highlight the beneficial effect of a physically active lifestyle in counteracting aberrant cancer-related gene expression programs by improving the redox buffering capacity.

MicroRNA-100 Enhances Autophagy and Suppresses Migration and Invasion of Renal Cell Carcinoma Cells via Disruption of NOX4-Dependent mTOR Pathway.

Renal cell carcinoma (RCC) is the most common kidney malignancy and has a poor prognosis owing to its resistance to chemotherapy. Recently, microRNAs (miRNAs or miRs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer. In the present study, we aim to explore the potential role of miR‐100 in RCC by targeting nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) through the mammalian target of rapamycin (mTOR) pathway. Initially, microarray‐based gene expression profiling of RCC was used to identify differentially expressed genes. Next, the expression of miR‐100 and NOX4 was examined in RCC tissues and cell lines. Then, the interaction between miR‐100 and NOX4 was identified using bioinformatics analysis and dual‐luciferase reporter assay. Gain‐of‐function or loss‐of‐function approaches were adopted to manipulate miR‐100 and NOX4 in order to explore the functional roles in RCC. The results revealed the presence of an upregulated NOX4 and a downregulated miR‐100 in both RCC tissues and cell lines. NOX4 was verified as a target of miR‐100 in cells. In addition, overexpression of miR‐100 or NOX4 silencing could increase autophagy while decreasing the expression of mTOR pathway‐related genes and migration and invasion. Conjointly, upregulated miR‐100 can potentially increase the autophagy and inhibit the invasion and migration of RCC cells by targeting NOX4 and inactivating the mTOR pathway, which contributes to an extensive understanding of RCC and may provide novel therapeutic options for this disease.

Signature genes associated with immunological non-responsiveness to anti-retroviral therapy in HIV-1 subtype-c infection.

ObjectiveHIV-infected individuals undergoing therapy may show an immunological-discordant response to therapy, with poor CD4+ T cells recovery, despite viral suppression below the detection limit. The present study was carried out to delineate the underlying molecular mechanisms of immunological non-responsiveness to HIV therapy.DesignWe conducted microarray-based whole gene expression profiles of 30 subjects infected with HIV-1 subtype C, in peripheral blood to discern the signature genes associated with immunological non-responsiveness. After a thorough analysis and comparison of gene-expression profiles, microarray data was validated via qRT-PCR approach.ResultsOverall, we found 10 genes significantly up-regulated and 60 genes down-regulated (≥2-fold change) in immunological non-responders as compared to responders. Based on these results and pathway analysis of the protein-protein interaction, 20 genes were shortlisted for validation in human infected cases. We found statistically significant differences in expression levels of twelve genes IL-1α, IL-1β, IL-7R, TNF-α, FoxP3, PDCD5, COX7B, SOCS1, SOCS3, RPL9, RPL23, and LRRN3 respectively among immunological non-responders compared to therapy responders, confirming their an intimate relationship with immunological responsiveness to therapy.ConclusionsAltogether, microarray and qRT-PCR validation results indicated that the aberrant expression of key genes involved in the regulation of T cell homeostasis, immune activation, inflammatory cytokine production, apoptosis, and immune-regulatory processes are possibly associated with immunological non-responsiveness in HIV-1 C infected individuals on ART.

The tumor-suppressive role of microRNA-873 in nasopharyngeal carcinoma correlates with downregulation of ZIC2 and inhibition of AKT signaling pathway.

Cancer stem cells (CSCs) are responsible for tumor initiation, relapse, and metastasis. Thus, residual CSCs after chemotherapy may result in poor prognosis for nasopharyngeal carcinoma (NPC). Emerging evidence suggests that differentially expressed microRNAs (miRNAs) regulate genes that carry out important functions in CSCs. Here we investigate the interaction of microRNA-873 (miR-873) with the Zic family member 2 (ZIC2) and the effects on downstream serine-threonine protein kinase (AKT) signaling pathway in CSCs in the context of NPC. Initially, microarray-based gene expression profiling identified ZIC2 as a key differentially expressed gene in NPC, which was subsequently confirmed to be upregulated in clinical NPC tissue samples. NPC cells were subjected to sphere-formation conditions in low-attachment plates, followed by sorting of CD133+ cells, which were selected as NPC stem cells after further characterization of stem cell biomarkers. ZIC2 was then shown to be enriched in NPC stem cells at both mRNA and protein levels. However, loss of ZIC2 was associated with the self-renewal, proliferative and tumorigenic properties of NPC stem cells. Next, miRNAs potentially able to target ZIC2 were predicted by the intersection of mirDIP and TargetScan database results, and miRNA miR-873 was found to be downregulated in NPC tissues in general but especially in NPC stem cells. Upregulation of miR-873 inhibited the stem-like properties and tumorigenicity of NPC stem cells, which was found to take place through downregulation of ZIC2 and disruption of the AKT signaling pathway. Collectively, the results obtained suggest that overexpression of miR-873 could aid NPC tumor suppression through reduction of the malignant potential of CSCs.

Transcription factor HIF1α promotes proliferation, migration, and invasion of cholangiocarcinoma via long noncoding RNA H19/microRNA-612/Bcl-2 axis

Cholangiocarcinoma, which is the most common invasive malignant tumor of the biliary tract, has poor prognosis. There is evidence suggesting that hypoxia-inducible factor 1α (HIF1α) plays an important role in cholangiocarcinoma. Also, microRNA-612 (miR-612) is another key regulator of cholangiocarcinoma. In this study, we investigate the scantly documented interaction of HIF1α and miR-612 in cholangiocarcinoma. We first undertook microarray-based cholangiocarcinoma gene expression profiles to screen out the differentially expressed long noncoding RNAs (lncRNAs) and genes. We used reverse transcription quantitative polymerase chain reaction to detect the expression of HIF1α in normal bile duct and cholangiocarcinoma tissues, and in corresponding cells lines. Cell counting kit 8, scratch, and Transwell assays were used to detect the proliferation, migration and invasion of cholangiocarcinoma cells. Cell cycle distribution and apoptosis were detected by flow cytometry. ChIP, dual luciferase reporter gene assay, RNA pull-down, and RNA immunoprecipitation were used to verify relationship between HIF1α and lncRNA H19, and lncRNA H19 and miR-612. We also monitored tumor formation in nude mice to verify the effect of HIF1α on cholangiocarcinoma. HIF1α expression was elevated in cholangiocarcinoma tissues and cells. Silencing HIF1α reduced proliferation, migration, and invasion of cholangiocarcinoma cells. HIF1α transcriptionally activated the expression of lncRNA H19. Overexpression of miR-612 could rescue the proliferation, migration and invasion of cholangiocarcinoma cells caused by lncRNA H19 overexpression. Taken together, HIF1α activated lncRNA H19-mediated miR-612/Bcl-2 pathway to promote cholangiocarcinoma, suggesting a promising therapeutic target for cholangiocarcinoma.

DNA and RNA next generation sequencing for personalizing cancer treatment: A single-center experience.

e13509 Background: Personalized multidrug therapies improve outcomes in patients with drug resistant cancer. For 8 years we have been using microarray-based gene expression profiling (360 procedures) to improve drug selection (Rebollo J et al, 2017; Sureda M et al, 2018). Recently, we have adopted Ion Torrent DNA and RNA Next Generation Sequencing (NGS) to improve the target detection ability. Methods: Since March 2018 DNA (95 analyses) and whole trancriptome RNA (78 analyses) NGS have been performed using Ion Torrent GeneStudio S5 System in fresh-frozen (RNA and DNA sequencing) and paraffin-embedded (DNA sequencing) biopsies obtained from tru-cut or surgical excision procedures from patients with metastatic cancer. Favourable chemotherapy drug profile, treatable (available inhibitory drugs) targets and potentially favourable immunologic signature have been investigated. Results: All biopsies were valid for DNA sequencing but in five patients were invalid (less than 30% viable tumor cells required) for RNA sequencing. Combined DNA and RNA sequencing have been studied in sixty patients with advanced cancer biopsies. Histologies have been NSCLC (12 patents), Breast (9), STS (6), NHL (4), NET (4), Pancreas (4), CRC (4), Ovary/Fallopian tube (3), Prostate (2), Cholangiocarcinoma (2), H&N (2), Esophagus, Bladder, Uterus, Melanoma, SCLC, Anal, Kidney and Cervix (1 each). Most of the patients had been previously treated with systemic therapies. Biopsies have been taken from Lymph nodes (18 patients), Liver (16), Soft Tissues (14), Lung (8), Peritoneum (3) and Adrenal Gland (1). RNA sequencing has predicted favourable chemotherapy drug profile (Median 3 drugs, Range 1-9) in 54 (90%) patients. In addition, treatable targets (Median 3 targets, Range 1-6) have been found in 40 (67%) patients. Favourable immunologic signature has been found in 22 (37%) patients. DNA sequencing has shown treatable (drug available) targets in 14 (23%) patients. In 12 (20%) patients, no targets have been found in any of the sequencing protocols (DNA and RNA). Conclusions: Tumor RNA and DNA sequencing provide potential useful information in personalized cancer treatment decision in a vast majority of advanced cancer patients independent of treatment status.

White tea and its active polyphenols lower cholesterol through reduction of very-low-density lipoprotein production and induction of LDLR expression

Emerging in vivo and vitro data suggest that white tea extract (WTE) is capable of favourably modulating metabolic syndrome, especially by ameliorating abnormal lipid metabolism. Microarray-based gene expression profiling was performed in HepG2 cells to analyze the effects of WTE from a systematic perspective. Gene Ontology and pathway analysis revealed that WTE significantly affected pathways related to lipid metabolism. WTE significantly downregulated apolipoprotein B (APOB) and microsomal triglyceride transfer protein (MTTP) expression and thereby reduced the production of very-low-density lipoprotein. In the meanwhile, WTE stimulated low-density lipoprotein-cholesterol (LDL-c) uptake through targeting low-density lipoprotein receptor (LDLR), as a consequence of the activation of sterol regulatory element-binding protein 2 (SREBP2) and peroxisome proliferator-activated receptor δ (PPARδ). Furthermore, WTE significantly downregulated triglycerides synthetic genes and reduced intracellular triglycerides accumulation. Besides, we demonstrated that the tea catechins epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) are abundant in WTE and contribute to the regulation of cholesterol metabolism related genes, including LDLR, MTTP and APOB. Our findings suggest white tea plays important roles in ameliorating abnormal lipid metabolism in vitro.

MicroRNA-212-3p regulates early neurogenesis through the AKT/mTOR pathway by targeting MeCP2

Compelling evidence has implicated role of microRNAs (miRNAs) in neurogenesis. Methyl-CpG Binding Protein 2 (MeCP2) was a key contributor to neurological disease. This study investigated whether miR-212-3p affects early neurogenesis associated with MeCP2. Microarray-based gene expression profiling of neurogenesis was employed to identify differentially expressed genes. Next, miR-212-3p expression in neural progenitor cells (NPCs) was detected using in situ hybridization and immunofluorescence. Effect of miR-212-3p and MeCP2 on cell viability, β-tubulin III expression and the AKT/mammalian target of rapamycin (mTOR) pathway activity was examined with gain- and loss-of-function experiments. In vivo experiments were also performed to verify effects of miR-212-3p on nerve tube development. MiR-212-3p expression was decreased while MeCP2 expression was increased during differentiation of NPCs. MiR-212-3p targets MeCP2 and down-regulates its expression, which resulted in repressed cell differentiation, proliferation as well as blocked AKT/mTOR pathway activation, subsequently early neurogenesis was prevented. Furthermore, overexpression of miR-212-3p inhibited nerve tube development in vivo. Taken together, miR-212-3p could restrain early neurogenesis through the blockade of AKT/mTOR pathway activation by targeting MeCP2, suggesting a promising therapeutic target for neurogenic disorders.

Long noncoding RNA LINC00324 exerts protumorigenic effects on liver cancer stem cells by upregulating fas ligand via PU box binding protein.

Hepatocellular carcinoma (HCC) represents a major cause of cancer death, but the molecular mechanism for its development has not yet been well characterized. Long noncoding RNAs (lncRNAs) are involved in a wide range of biological processes via their roles as oncogenes or tumor suppressor genes. The present study aimed to elucidate the role of LINC00324 in HCC through its interaction with Fas ligand (FasL). Initially, microarray-based gene expression profiling of HCC was employed to identify differentially expressed genes. Next, the expression of LINC00324 in HCC tissues and liver cancer stem cell (LCSC) lines was examined using RT-qPCR. Then, the interaction among LINC00324, PU box binding protein (PU.1) and FasL was identified with RIP, ChIP and dual-luciferase reporter gene assays. The effect of LINC00324 on viability, proliferation, migration, invasion, and apoptosis as well as the tumorigenesis of transfected cells was examined with gain- and loss-of-function experiments. LINC00324 and FasL were highly expressed in HCC. LINC00324 regulated FasL expression via interaction with PU.1. Silencing of LINC00324 or FasL suppressed expression of stemness-related genes, cell viability, proliferation, migration, invasion, self-renewal, and tumorigenesis, but enhanced cell apoptosis. Taken together, LINC00324 promotes the expression of FasL through the recruitment of PU.1, which ultimately maintains the biological properties of LCSCs, thus, highlighting LINC00324 as a promising therapeutic candidate for HCC.

RETRACTED ARTICLE: Efficient attribute selection technique for leukaemia prediction using microarray gene data

The recent advancements in today’s medical sciences regarding Data Analytics have made it possible for the use of efficient techniques for analysis. For prognosis, diagnosis and cancer treatment, a microarray-based gene expression profiling is considered. Informative genes causing cancer are determined through the deoxyribonucleic acid microarray technique. Dimensionality is the utmost concern while working with multi-dimensional data analysis which acts as a barrier in extracting information from a dataset which leads to costly computational complexity. Thus, an imperative task in the selection of relevant features in the analysis of cancer microarray datasets is crucial towards effective classification. This work focuses on variable selection techniques by utilizing effective correlation for attribute selection along with ant colony optimization. The criterion of a given classifier is maximized through wrapper-based attribute selection, and so it needs efficient searching techniques in finding optimal feature combinations. A new wrapper-based selection technique which uses ant lion optimization (ALO) in finding optimal feature set is proposed in this work which maximizes classification performance. The natural shooting procedure of ant lions is imitated in the proposed ALO algorithm. Support vector machine technique was utilized for the classification of chosen marker genes.

Upregulation of miR-101a Suppresses Chronic Renal Fibrosis by Regulating KDM3A via Blockade of the YAP-TGF-β-Smad Signaling Pathway

Renal fibrosis denotes a common complication of diabetic nephropathy and is a predominant cause of end-stage renal disease. Despite the association between microRNAs (miRNAs or miRs) and renal fibrosis, miRNAs have been reported to play a vital role in the development of chronic renal fibrosis. Therefore, the aim of the present study was to investigate the possible function of miR-101a in chronic renal fibrosis. Initially, microarray-based gene expression profiling of renal fibrosis was employed to screen the differentially expressed genes. An in vivo mouse model of chronic renal fibrosis induced by a unilateral ureteral obstruction (UUO) and an in vitro cell model induced by aristolochic acid (AA) were constructed. miR-101a expression was examined using a fluorescence in situ hybridization (FISH) assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then, the interaction between miR-101a and KDM3A was identified using an online website combined with a dual-luciferase reporter assay. Finally, gain- and loss-of-function experiments were conducted to elucidate the effect of miR-101a on the expression of Col1a1, fibronectin, α-smooth muscle actin (α-SMA), and YAP-TGF-β (transforming growth factor β)-Smad signaling pathway-related genes, as well as the degree of renal fibrosis. miR-101a was poorly expressed while KDM3A was robustly induced in chronic renal fibrosis tissues and cells. In addition, miR-101a could target and downregulate KDM3A expression, which led to elevated TGIF1, inhibited expression of Collagen I (Col1a1), fibronectin, α-SMA, YAP1, and TGF-β2 along with the extent of Smad2/3 phosphorylation, as well as delayed renal fibrosis degree. Besides, overexpressed YAP/TGF-β2 or inhibited TGIF1 partially restored the inhibitory effect of miR-101a on chronic renal fibrosis. Taken together, miR-101a could potentially slow down chronic renal fibrosis by the inactivation of the YAP-TGF-β-Smad signaling pathway via KDM3A, highlighting the potential of miR-101a as a therapeutic target for chronic renal fibrosis treatment.